Hemostasis involves control of bleeding and is typically viewed as a cascade of processes that help regulate blood coagulation. Central to the hemostasis process is thrombin generation. Thrombin is a proteolytic enzyme, which is generated in the blood following activation of the coagulation system as a result of proteolysis of prothrombin. It facilitates the clotting of blood by activating fibrinogen and releasing the fibrinopeptides A and B to form fibrin. Thrombin has many functions extending from coagulation activator and inhibitor to cellular regulator. Thrombin generation, essential in the hemostatic process and the maintenance of vascular integrity is also associated with vasoocclusive events leading to heart attacks, stroke, pulmonary embolism and venous thrombosis.
The physiologic pathway for thrombin generation occurs when the vascular system is perturbed. A perturbation is thought to facilitate the expression of tissue factor which binds circulating factor VIIa and forms the extrinsic tenase complex. During an initiation phase of this process, factor IX and factor X are converted to the serine proteases factor IXa and factor Xa, and the initial factor Xa formed, activates prothrombin to thrombin. See e.g., Mann, K. G. et al. (1990) Blood 76: 1; Mann, K. G. et al. (1992), supra.
Thrombin activates platelets, fibrinogen, factor XIII and the procofactors factor VIII and factor V providing the membrane receptors, factor VIIIa and factor Va. The membrane bound cofactors then are believed to bind factor IXa (the intrinsic tenase) and factor Xa (prothrombinase) to provide the major products of the hemostatic process, during a propagation phase of thrombin generation. As thrombin is being generated it is also down regulated by the stoichiometric inhibitors antithrombin III and tissue factor pathway inhibitor, and in the vasculature, the dynamic thrombin—thrombomodulin—protein C system.
On average, in tissue factor triggered whole blood in vitro, it is thought that approximately 60% of prothrombin is activated to thrombin, of which approximately 3% (10-20 nM thrombin-antithrombin III, TAT) produced during the initiation phase is required to clot whole blood. Following the visual clot time, 97% of the thrombin is generated during the propagation phase. In healthy individuals, the initiation phase duration and the activation of procoagulants during the propagation phase is believed to be fairly constant.
In hemophilia A the initiation phase is thought to be delayed, but the major defect observed is a severely attenuated propagation phase. The propagation phase is also depressed for individuals on coumadin therapy, in thrombocytopenic individuals, and by anti-platelet glycoprotein IIbIIIa therapies. These observations suggest that the combination of the generation of a sufficient level of thrombin (propagation phase), in a timely fashion (initiation phase), is of importance.
A thrombin assay that can measure the capacity of an individual to produce thrombin over time is needed. Such an assay would find use, for instance, in monitoring healthy individuals for variations in their thrombin levels potentially signifiying a change in hemostatis; as well the hemostatic competence of patients with hemophilia, individuals on anticoagulant therapy, thrombocytopenic individuals, and with patients on anti-platelet therapies, among other uses.
U.S. Pat. No. 6,403,381 to Mann et al. describes methods for inhibiting blood clotting which include adding corn trypsin inhibitor in an amount sufficient to inhibit blood clotting, in order to permit clotting assays featuring prolonged clotting times. This patent also discloses clotting assays using whole or minimally altered blood.